Selective production of organic acids in anaerobic acid reactor by pH control

The selective production of organic acids by anaerobic acidogenesis with pH control was examined using a chemostat culture. The results showed that the product spectrum in the acid reactor strongly depended on the culture pH. Under acidic and neutral conditions, the main products were butyric acid, while acetic and propionic acids were the main products under the basic condition. This phenomenon was reversible between the acidic and basic conditions, and was not affected by the dilution rate. The change in the main products was caused by the change in the dominant microbial populations, from butyric acid-producing bacteria to propionic acid-producing bacteria in the acid reactor due to the pH shift. The control of culture pH was considered to be a useful way for controlling the product spectrum in the anaerobic acid reactor.     

Continuous hydrolysis of tallow with immobilized lipase in a microporous membrane

Thermostable lipase from Thermomyces lanuginosus was immobilized in untreated microporous membranes. Melted tallow pumped through the membrane did not wash the enzyme out. From 0.4 to 0.9% of the soluble activity remained after immobilization with half-lives of 1-2 months or more at 50 degrees C. Membranes can be acid/base washed and reloaded with enzyme with no adverse effects. Buffer was required for a long half-life, and recycling the buffer improved the mass transfer of glycerol out of the immobilized lipase reactor. Immobilized activity was unaffected when the pH of the aqueous product changed from 5.5 to 6.5.     

Anaerobic treatment of fish meal process waste-water in a UASB reactor at high pH

Summary The influence of high pH on anaerobic degradation of fish process waste-water with a high total ammonia concentration was investigated in an upflow anaerobic sludge blanket (UASB) reactor. More than 99% of volatile fatty acids and trimethylamine in the process waste-water were degraded up to pH 7.9. Above pH 7.9 only the conversion of acetate was slightly decreased. At pH 8.3 serious disintegration of the granules occurred leading to process failure. Increasing the pH changed the physical characteristics of the granules leading to decreased density, size, and volatile solids content. After 4 months of acclimatization to high pH in the reactor, the specific methanogenic activity of the granular biomass was the same from pH 7.1 to 8.5. At pH 8.3 and 8.5, acclimatization had improved the specific activity by 25 and 50%, respectively. However, the acclimatized biomass generally showed a decreased activity (60%) at all pH values tested below the acclimatization pH.Offprint requests to: B. K. Ahring     

Rapid formation of hydrogen-producing granules in an anaerobic continuous stirred tank reactor induced by acid incubation

A novel approach to rapidly initiate granulation of hydrogen-producing sludge was developed in an anaerobic continuous stirred tank reactor at 37 degrees C. To induce microbial granulation, the acclimated culture was subject to an acid incubation for 24 h by shifting the culture pH from 5.5 to 2.0. The culture was resumed to pH 5.5 after the incubation and the reactor was operated at hydraulic retention times (HRTs) of 12, 6, 2, 1, and 0.5 h in sequence. Microbial aggregation took place immediately with the initiation of acid incubation and granules were developed at 114 h. No granule was observed in the absence of acid incubation in the control test. Changing the culture pH resulted in improvement in surface physicochemical properties of the culture favoring microbial granulation. The zeta potential increased from -11.6 to -3.5 mV, hydrophobicity in terms of contact angle improved from 31 degrees to 43 degrees and extracellular proteins/polysaccharides ratio increased from 0.2 to 0.5-0.8. Formation of granular sludge facilitated biomass retention of up to 32.2 g-VSS/L and enhanced hydrogen production. The hydrogen production rate and hydrogen yield increased with the reduction in HRT at an influent glucose concentration of 10 g/L once steady granular sludge layer was formed, achieving the respective peaks of 3.20 L/L x h and 1.81 mol-H(2)/mol-glucose at 0.5 h HRT. The experimental results suggested that acid incubation was able to initiate the rapid formation of hydrogen-producing granules by regulating the surface characteristics of microbial aggregates in a well-mixed reactor, which enhanced the hydrogen production.     

Study of the acidogenic phase of the anaerobic fermentation of stillage from ethanol distilleries

Summary A bench scale continuously stirred reactor was used to study the acidogenic phase of the anaerobic fermentation of stillage. The residence time of the effluent in the reactor ranged from 15.7 to 8.2 hours, pH was around 5.0 and temperature was maintained at 35°C. The results indicate that the residence time had no appreciable effect on the production or composition of the organic acids. The main acid products found in the reactor effluent were acetic, propionic and butiric acids.     

Extractive fermentation for butyric acid production from glucose by Clostridium tyrobutyricum

A novel extractive fermentation for butyric acid production from glucose, using immobilized cells of Clostridium tyrobutyricum in a fibrous bed bioreactor, was developed by using 10% (v/v) Alamine 336 in oleyl alcohol as the extractant contained in a hollow-fiber membrane extractor for selective removal of butyric acid from the fermentation broth. The extractant was simultaneously regenerated by stripping with NaOH in a second membrane extractor. The fermentation pH was self-regulated by a balance between acid production and removal by extraction, and was kept at approximately pH 5.5 throughout the study. Compared with conventional fermentation, extractive fermentation resulted in a much higher product concentration (>300 g/L) and product purity (91%). It also resulted in higher reactor productivity (7.37 g/L. h) and butyric acid yield (0.45 g/g). Without on-line extraction to remove the acid products, at the optimal pH of 6.0, the final butyric acid concentration was only approximately 43.4 g/L, butyric acid yield was 0.423 g/g, and reactor productivity was 6.77 g/L. h. These values were much lower at pH 5.5: 20.4 g/L, 0.38 g/g, and 5.11 g/L. h, respectively. The improved performance for extractive fermentation can be attributed to the reduced product inhibition by selective removal of butyric acid from the fermentation broth. The solvent was found to be toxic to free cells in suspension, but not harmful to cells immobilized in the fibrous bed. The process was stable and provided consistent long-term performance for the entire 2-week period of study.     

Formation of gluconic acid at low pH-values by free and immobilized Aspergillus niger cells during citric acid fermentation

Summary A recently developed immobilization method, characterized by the adsorption of the mycelia onto a glass-carrier in a fixed-bed reactor, was applied for citric acid production by Aspergillus niger ATCC 9142, and compared with conventional culture techniques.In a fixed-bed reactor and in a stirred fermenter a rapid gluconic acid production started immediately after nitrate exhaustion, though the pH was below 2.5 During a second production phase a comparatively small amount of citric acid was formed.In surface and shaken-flask cultures nearly no gluconic acid could be found, whereas citric acid yields were significantly higher than in the fixed-bed reactor and in the stirred fermenter.Manganese (0.8×10^–7 Mol×dm^–3 after 6 days incubation) from the stainless steel parts of the vessel seemed to be responsible for both gluconic acid production and small citric acid yields in the stirred fermenter and in the fixed-bed reactor.     

Effect of pH on phase separated anaerobic digestion

A pilot scale experiment was performed for a year to develop a two-phase anaerobic process for piggery wastewater treatment (COD: 6,000 mg/L, BOD: 4,000 mg/L, SS: 500 gm/L, pH 8.4, alkalinity 6,000 mg/L). The acidogenic reactor had a total volume of 3 m³, and the methanogenic reactor, an, anaerobic up-flow sludge filter, combining a filter and a sludge bed, was also of total volume 3 m³ (1.5 m³ of upper packing material). Temperatures of the acidogenic and methanogenic reactors kept at 20°C and 35°C., respectively. When the pH of the acidogenic reactor was controlled at 6.0–7.0 with HCl, the COD removal efficiency increased from 50 to 80% over a period of six months, and as a result, the COD of the final effluent fell in the range of 1,000–1,500 mg/L. BOD removal efficiency over the same period was above 90%, and 300 to 400 mg/L was maintained in the final effluent. The average SS in the final effluent was 270 mg/L. The methane production was 0.32 m³ CH₄/kg COD_removed and methane content of the methanogenic reactor was high value at 80–90%., When the pH of the acidogenic reactor was not controlled over the final two months, the pH reached 8.2 and acid conversion decreased compared with that of pH controlled, while COD removal was similar to the pH controlled operation. Without pH control, the methane content in the gas from methanogenic reactor improved to 90%, compared to 80% with pH control.     

Hydrogen bio-production through anaerobic microorganism fermentation using kitchen wastes as substrate

In order to treat the kitchen wastes and produce hydrogen, anaerobic fermentation technology was used in this experiment. The results showed that the fermentation type changed from mixed acid fermentation to ethanol fermentation in a continuous stirred tank reactor (CSTR) 22 days after start-up. The maximum efficiency of hydrogen bio-production in the CSTR was 4.77 LH₂/(L reactor d) under the following conditions: organic loading rate (OLR) of 32–50 kg COD/(m³ d), oxidation reduction potential (ORP) of −450 to −400 mV, influent pH value of 5.0–6.0, effluent pH value of 4.0–4.5, influent alkalinity of 300–600 mg/l, temperature of 35 ± 1°C and hydraulic retention time (HRT) of 7 h. An artificial neural network (ANN) model was established, and each parameter influencing the performance of the reactor was compared using the method of partitioning connection weights (PCW). The results showed that OLR, pH, ORP and alkalinity could influence the fermentation characteristics and hydrogen yield of the anaerobic activated sludge; with an influence hierarchy: OLR > pH values > ORP > alkalinity. An economic analysis showed that the cost of producing hydrogen in this experiment was less than the cost of electrolysis of water.     

Copper removal from an industrial waste by bioleaching

Summary Copper contained in a solid industrial waste produced in a silicone manufacturing process was leached with spent iron medium from aThiobacillus ferrooxidans culture. Most effective leaching was observed in a continuously fed, dual reactor system. Spent iron medium was generated by growingT. ferrooxidans in 0.9 K iron medium at pH 1.5 in the first reactor, and was transferred to a second reactor in which it leached the copper from the waste. Leaching was effective at a pulp density of the waste material as high as 20%. In experiments run at a pulp density of 2.5%, the spent iron medium was most efficient in leaching copper when it was first diluted 100-fold with a mineral salts solution at pH 1.5. Removal of the copper from the waste appeared to involve its displacement by acid, dissolved mineral salts, and ferric iron. Potentials for practical application of this process are discussed.     

Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of D-amino acid oxidase and pyruvate oxidase

A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA). The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala. The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction. The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala. It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1). A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM. The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala. The D-Ala content in some fish sauces was also determined using the proposed sensor system. The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method. From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx).     

Application of a fuzzy logic control system for continuous anaerobic digestion of low buffered, acidic energy crops as mono-substrate

A fuzzy logic control (FLC) system was developed at the Hamburg University of Applied Sciences (HAW Hamburg) for operation of biogas reactors running on energy crops. Three commercially available measuring parameters, namely pH, the methane (CH4) content, and the specific gas production rate (spec. GPR = m(3)/kg VS/day) were included. The objective was to avoid stabilization of pH with use of buffering supplements, like lime or manure. The developed FLC system can cover most of all applications, such as a careful start-up process and a gentle recovery strategy after a severe reactor failure, also enabling a process with a high organic loading rate (OLR) and a low hydraulic retention time (HRT), that is, a high throughput anaerobic digestion process with a stable pH and CH4 content. A precondition for a high load process was the concept of interval feeding, for example, with 8 h of interval. The FLC system was proved to be reliable during the long term fermentation studies over 3 years in one-stage, completely stirred tank reactors (CSTR) with acidic beet silage as mono-input (pH 3.3-3.4). During fermentation of the fodder beet silage (FBS), a stable HRT of 6.0 days with an OLR of up to 15 kg VS/m(3)/day and a volumetric GPR of 9 m(3)/m(3)/day could be reached. The FLC enabled an automatic recovery of the digester after two induced severe reactor failures. In another attempt to prove the feasibility of the FLC, substrate FBS was changed to sugar beet silage (SBS), which had a substantially lower buffering capacity than that of the FBS. With SBS, the FLC accomplished a stable fermentation at a pH level between 6.5 and 6.6, and a volatile fatty acid level (VFA) below 500 mg/L, but the FLC had to interact and to change the substrate dosage permanently. In a further experiment, the reactor temperature was increased from 41 to 50 degrees C. Concomitantly, the specific GPR, pH and CH4 dropped down. Finally, the FLC automatically enabled a complete recovery in 16 days.     

Modeling of two-phase anaerobic process treating traditional Chinese medicine wastewater with the IWA Anaerobic Digestion Model No. 1

The aim of the study was to implement a mathematical model to simulate two-phase anaerobic digestion (TPAD) process which consisted of an anaerobic continuous stirred tank reactor (CSTR) and an upflow anaerobic sludge blanket (UASB) reactor in series treating traditional Chinese medicine (TCM) wastewater. A model was built on the basis of Anaerobic Digestion Model No. 1 (ADM1) while considering complete mixing model for the CSTR, and axial direction discrete model and mixed series connection model for the UASB. The mathematical model was implemented with the simulation software package MATLABTM/Simulinks. System performance, in terms of COD removal, volatile fatty acids (VFA) accumulation and pH fluctuation, was simulated and compared with the measured values. The simulation results indicated that the model built was able to well predict the COD removal rate (−4.8–5.0%) and pH variation (−2.9–1.4%) of the UASB reactor, while failed to simulate the CSTR performance. Comparing to the measured results, the simulated acetic acid concentration of the CSTR effluent was underpredicted with a deviation ratios of 13.8–23.2%, resulting in an underprediction of total VFA and COD concentrations despite good estimation of propionic acid, butyric acid and valeric acid. It is presumed that ethanol present in the raw wastewater was converted into acetic acid during the acidification process, which was not considered by the model. Additionally, due to the underprediction of acetic acid the pH of CSTR effluent was overestimated.     

Effects of key operational parameters on biohydrogen production via anaerobic fermentation in a sequencing batch reactor

In this study, a series of tests were conducted in a 6 L anaerobic sequencing batch reactor (ASBR) to investigate the effect of pH, hydraulic retention time (HRT) and organic loading rate on biohydrogen production at 28 °C. Sucrose was used as the main substrate to mimic carbohydrate-rich wastewater and inoculum was prepared from anaerobic digested sludge without pretreatment. The reactor was operated initially with nitrogen sparging to form anaerobic condition. Results showed that methanogens were effectively suppressed. The optimum pH value would vary depending on the HRT. Maximum hydrogen production rate and yield of 3.04 L H₂/L reactor d and 2.16 mol H₂/mol hexose respectively were achieved at pH 4.5, HRT 30 h, and OLR 11.0 kg/m³ d. Two relationships involving the propionic acid/acetic acid ratio and ethanol/acetic acid ratio were derived from the analysis of the metabolites of fermentation. Ethanol/acetic acid ratio of 1.25 was found to be a threshold value for higher hydrogen production.     

Propionic acid accumulation and degradation during restart of a full-scale anaerobic biowaste digester

The methane formation rate of 300 m(3) of sludge from a full scale biowaste reactor, that was stored without feeding for six weeks during a maintenance period, was about 60% of the methanogenic activity before maintenance. The 300 m(3) sludge was then pumped back into the biowaste reactor. On the third day, after refilling of the stored biowaste suspension, anaerobic conditions were obtained and feeding was started by addition of 36.1 m(3) of fresh biowaste suspension (=11.3 tons biowaste). The pH dropped from originally pH 7.7 to pH 7.3 and later on to pH 6.8, which was considered the minimum allowed pH for methanogenesis to recover. Maximum concentrations of acetate (1.78 gl(-1)), n-butyrate (0.57 gl(-1)) and n-valerate (0.44 gl(-1)) accumulated during the following days with feeding of 11.8 tons on day 5 and twice 6.5 tons on days 7 and 9, respectively. Thereafter, acetate, n-butyrate and n-valerate were degraded completely, whereas the concentration of propionate was still increasing. Propionic acid was the dominant fatty acid during the restart period and reached its maximum concentration of 6.2 gl(-1) 17 days after start of feeding. This high level of propionate was degraded completely in about 5 days with maximum degradation rates of 2.14 gl(-1)d(-1), and the pH of the anaerobic sludge increased from 7.1 to 7.4. During restart, the methane content of the biogas increased successively to 65%. Samples that were taken at different time intervals during the restart phase of the methane reactor showed different fatty acid degradation capabilities. After 10 days, when acetate and n-butyrate still accumulated in the methane reactor the maximum acetate degradation rate was 1.52 gl(-1)d(-1) and the n-butyrate degradation rate was 0.51 gl(-1)d(-1). Oxidation of n-valerate caused an increase of propionate, which was degraded after a lag phase of 6 days with a maximum rate of 0.6 gl(-1)d(-1). In the samples taken after 16 and 23 days, the propionate degradation rate increased to 1.42 gl(-1)d(-1) and 1.55 gl(-1)d(-1), respectively, and the lag phase for propionate degradation was reduced or had disappeared completely. The maximum propionate degradation rate was measured in the methane reactor in the fourth week after restart. The synthrophic propionate oxidizing bacteria were apparently the most suffering bacteria during sludge storage. If the propionate oxidizing bacteria could be kept active and the propionate degrading activity of the biowaste suspension of 6.16 gl(-1)d(-1) before the maintenance period could be maintained, then accumulation of 6.2 gl(-1) propionate in the methane reactor after restart could be avoided and full activity reached even earlier.     

Propionic acid fermentation of lactose by Propionibacterium acidipropionici: effects of pH

Batch propionic acid fermentation of lactose by Propionibacterium acidipropionici were studied at various pH values ranging from 4.5 to 7.12. The optimum pH range for cell growth was between 6.0 and 7.1, where the specific growth rate was approximately 0.23 h(-1). The specific growth rate decreased with the pH in the acids have been identified as the two major fermentation products from lactose. The production of propionic acid was both growth and nongrowth associated, while acetic acid formation was closely associated with cell growth. The propionic acid yield increased with decreasing pH; It changed from approximately 33% (w/w) at pH 6.1-7.1 to approximately 63% at pH 4.5-5.0. In contrast, the acetic acid yield was not significantly affected by the pH; it remained within the range of 9%-12% at all pH values. Significant amounts of succinic and pyruvic acids were also formed during propionic acid fermentation of lactose. However, pyruvic acid was reconsumed and disappeared toward the end of the fermentation. The succinic acid yield generally decreased with the pH, from a high value of 17% at pH 7.0 to a low 8% at pH 5.0 Effects of growth nutrients present in yeast ex-tract on the fermentation were also studied. In general, the same trend of pH effects was found for fermentations with media containing 5 to 10 g/L yeast extract. However, More growth nutrients would be required for fermentations to be carried out efficienytly at acidic pH levels.     

Biochemical studies on an acidophilic, thermophilic bacterium, Bacillus acidocaldarius: isolation of bacteria, intracellular pH, and stabilities of biopolymers.

Acidophilic, thermophilic bacteria were isolated from Japanese acidic hot springs. They were spore-forming rods, identified as Bacillus acidocaldarius. DNA extracted from these acido-thermophiles showed no abnormality in chemical structure; it was instantly denatured and gradually decomposed giving rise to apurinic acid in a hot acid environment milder than the optimal conditions for the growth of the acido-thermophiles. Glyceraldehyde-3-phosphate dehydrogenase extracted from B. acidocaldarius was not active at pH 5 or less, and was resistant to heat at neutral but not acid pH. The intracellular pH was computed to be neutral by using dimethyl-2,4-oxazolidinedione. When uncouplers or inhibitors of respiration were added to the cells suspended in hot acid solution, the estimated pH was not changed and glyceraldehyde-3-phosphate dehydrogenase in the cells was not denatured. These results suggest that the cytoplasm of B. acidocaldarius is a hot neutral environment, and that a pH gradient across the cell envelope can be maintained even when oxidative phosphorylation or respiration is inhibited.     

An integral dynamic model for the UASB reactor

In this article a dynamic model of a continuous working UASB reactor is described. It results from the integration of the fluid flow pattern in the reactor, the kinetic behavior of the bacteria (where inhibition and limitation were taken into account), and the mass transport phenomena between different compartments and different phases. The mathematical equations underlying the model and describing the important mechanisms were programmed and prepared for computations and simulations by computer. The settler efficiency has to be over 99% to prevent the reactor from wash-out. When the settler efficiency is over 99%, the total sludge content of the reactor increases steadily, so the reactor is hardly ever in a steady state. This implies dynamic modeling. The model is able to predict the various observable and nonobservable or difficult to observe state variables, e.g., the sludge bed height, the sludge blanket concentration, the short-circuiting flows over bed and blanket, and the effluent COD concentration as a function of the hydrodynamic load, COD load, pH, and settler efficiency. The optimal pH value is between 6.0 and 8.0; fatty acid shock loadings are difficult to handle outside this optimal pH range.     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang